The overall objective of the proposed research is to understand processes and mechanisms involved in the organization and distribution of proteins and lipids in the plasma membrane of both resting and receptor activated cells. As model systems, we will focus on G-protein coupled receptors (GPCRs) and integrins. Chemokine/chemoattractant GPCRs, such as the N-Formyl peptide receptor (FPR), regulate numerous aspects of leukocyte function, including controlling hematopoiesis, chemotaxis and activation as in the event of physiological insult. GPCR activation has been suggested to lead to a redistribution of receptor into rafts in the plasma membrane. This activation also leads to alterations in the functions of integrins, resulting in clustering and conversion to a high affinity state. Integrin clustering has been suggested to be a critical determinant in regulating the avidity of its binding interactions and hence its physiological function. Thus, both the organization of the protein within the membrane (clustering) and the distribution of lipids about the protein (rafts) are likely to represent critical determinants in the proper functioning of membrane proteins. Although rafts are widely believed to play a critical role in mediating biological functions, it has proven exceedingly difficult to provide a robust characterization of rafts in terms of size, composition, and dynamics. It is noted that chemical composition of membranes is dynamic due to trafficking of vesicles, metabolism of lipids and insertion and removal of lipid components. We propose to use a combination of spectroscopic techniques to image real time changes in the lateral organization of lipid membranes in live cells by measuring energy transfer between fluorescently-labeled lipid probes and receptors bound to fluorescently labeled small molecule ligands. The data will be analyzed in terms of distances of closest approach between donors (on lipid probes or bound to proteins) and acceptors (on lipid probes or proteins). Distance of closest approach in membranes reflect the size of a protein, or conformation (integrins), the presence of boundary lipid which excludes the acceptor, or the distance of the donor above the membrane. The aims are: Aim 1. To characterize the structure function relationship associated with integrin activation and connection to membrane organization. Aim 2. To assess in real time the relationship between GPCR signaling and membrane reorganization. Aim 3. To generate integrin GFP chimerae and cytoplasmic tail alpha/L and beta2 mutants of alpha4beta1 and assess lateral organization in K562 cells. The proposed work provides a unique training opportunity to use biophysical tools and concepts combined with molecular and cell biological approaches to address problems of immense interest to cell biology. Completion of the proposed work will yield a better mechanistic grasp of the dynamics of membrane organization as a factor in signaling and cell adhesion.